Plants having changed development and a method for making the same

ABSTRACT

A method to change development of a plant or plant part, when compared to the wild-type plant or plant part, by increasing or decreasing expression in a plant or plant part of a cdc27a nucleic acid sequence and/or increasing or decreasing levels and/or activity in a plant of a CDC27A protein; a plant obtained by this method; a genetic construct for enacting the method; a food including the plant or plant part; an enzyme or pharmaceutical produced by the plant or plant part; and a method of making an enzyme or pharmaceutical by the plant or plant part.

The present invention concerns a method for changing development of aplant. More specifically, the present invention concerns a method forchanging plant development by increased or decreased expression of acdc27a nucleic acid and/or by increased or decreased activity and/orlevels of a CDC27A protein in a plant. The present invention alsoconcerns plants having increased or decreased expression of a cdc27anucleic acid and/or increased or decreased activity and/or levels of aCDC27A protein, which plants have changed development relative tocorresponding wild type plants.

The ever-increasing world population and the dwindling supply of arableland available for agriculture fuel agricultural research towardsimproving the efficiency of agriculture. Conventional means for crop andhorticultural improvements utilise selective breeding techniques toidentify plants having desirable characteristics. However, suchselective breeding techniques have several drawbacks, namely that thesetechniques are typically labour intensive and result in plants thatoften contain heterogeneous genetic components that may not alwaysresult in the desirable trait being passed on from parent plants.Advances in molecular biology have allowed mankind to modify thegermplasm of animals and plants. Genetic engineering of plants entailsthe isolation and manipulation of genetic material (typically in theform of DNA or RNA) and the subsequent introduction of that geneticmaterial into a plant. Such technology has the capacity to deliver cropsor plants having various improved economic, agronomic or horticulturaltraits. A trait of particular economic interest is yield. Yield isnormally defined as the measurable produce of economic value from acrop. This may be defined in terms of quantity and/or quality. Cropyield is influenced by the typical stresses to which plants or crops aresubjected. Such stresses include environmental (abiotic) stresses (suchas temperature stresses caused by atypical high or low temperatures;stresses caused by nutrient deficiency; stresses caused by lack of water(drought)) and biotic stresses (which can be imposed on plants by otherplants (weeds), animal pests and pathogens). Crop yield may not only beincreased by combating one or more of the stresses to which the crop orplant is subjected, but may also be increased by modifying the inherentgrowth and development mechanisms of a plant.

The inherent growth mechanisms of a plant reside in a highly orderedsequence of events collectively known as the ‘cell cycle’. Progressionthrough the cell cycle is fundamental to the growth of the organisms andis crucial to cell proliferation. The major components of the cell cycleare highly conserved in yeast, mammals and plants. The cell cycle istypically divided into the following sequential phases: G0-G1-S-G2-M.DNA replication or synthesis generally takes place during the S phase(“S” is for DNA synthesis) and mitotic segregation of the chromosomesoccurs during the M phase (the “M” is for mitosis), with intervening gapphases, G1 (during which cells grow before DNA replication) and G2 (aperiod after DNA replication during which the cell prepares fordivision). Cell division is completed after cytokinesis, the last stepof the M phase. Cells that have exited the cell cycle and that havebecome quiescent are said to be in the G0 phase. Cells in this phase canbe stimulated to re-enter the cell cycle at the G1 phase. The “G” in G1,G2 and G0 stands for “gap”. Completion of the cell cycle process allowseach daughter cell during cell division to receive a full copy of theparental genome.

Cell division is controlled by two principal cell cycle events, namelyinitiation of DNA synthesis and initiation of mitosis. Each transitionto each of these key events is controlled by a checkpoint represented byspecific protein complexes (involved in DNA replication and division).The transition between the different phases of the cell cycle, andtherefore progression through the cell cycle, is driven by the formationand activation of different heterodimeric serine/threonine proteinkinases, generally referred to as cyclin-dependent kinases (CDKs).Progression trough the cell cycle involves alternating phases of highand low activity of cyclin-dependent kinases. The anaphase-promotingcomplex (APC) is a multisubunit ubiquitin ligase triggering proteolyticdestruction of mitotic cyclins and is an important regulator of thelow-activity phase of cyclin dependent kinases. Cdc27 has been describedas a member of the APC complex, which is involved in the degradation ofmitotic cyclins during of the cell cycle to promote the anaphase ofmitosis.

The inherent development mechanisms of a plant reside in sequence ofevents leading to cell differentiation, which is crucial for thefunction of a multicellular organism. The meristem regions of higherplants contain cells with high mitotic activity and these regionscontinuously produce new cells. Once departed from the meristem, thecells expand and fully differentiate. This differentiation continueswhen mitotic activity ceases, so that plant development can proceed.

The ability to influence the differentiation and development in a plant(either using recombinant DNA technology or using non-recombinantmeans), and to thereby modify various developmental characteristics of aplant, would have many applications in areas such as crop enhancement,plant breeding, production of ornamental plants, aboriculture,horticulture and forestry.

The isolation and characterization of a cdc27a gene from Arabidopsisthaliana was described in international patent application WO0102430. InWO0102430 there is disclosed the use of cdc27a muteins or thedown-regulation of cdc27 to cause a malfunction of the APC complex andto cause endoreduplication via stimulation of DNA synthesis and/orblockage of mitosis. This document describes the link between cdc27A andDNA synthesis and/or mitosis and the use of cdc27a genes, proteins orinactivated variants/muteins in a plants, to influence processesinvolving DNA synthesis and/or mitosis such as DNA replication, celldivision and endoreduplication.

It has now been found that increasing or decreasing expression in aplant of a cdc27a nucleic acid and/or increasing or decreasing activityand/or levels in a plant of a CDC27A protein gives plants havingaccelerated development. Since plant differentiation and development areprocesses occurring after DNA synthesis and cell division, it wassurprising to find that these processes were influenced by the cdc27atransgene. More particularly, the effects of the cdc27a transgene wereaccelerated rate of development, increased of size and/or number oforgans and early flowering, which processes are based on differentiationof the cells and developmental patterns rather than on DNA synthesis andcell division.

Therefore according to a first embodiment of the present invention,there is provided a method to change development of a plant or plantpart compared to the wild-type plant or plant part, which methodcomprises increasing or decreasing expression in a plant of a cdc27anucleic acid sequence and/or increasing or decreasing levels and/oractivity in a plant of a CDC27A protein.

Increasing or decreasing expression of a cdc27a nucleic acid and/orincreasing or decreasing of the activity and/or levels of a CDC27Aprotein encompasses changed expression of a gene and/or changed activityand/or levels of a gene product, namely a polypeptide, in specific cellsor tissues. The changed expression, activity and/or levels is changedcompared to expression, activity and/or levels of a cdc27a gene orprotein in corresponding wild-type plants. The changed gene expressionmay result from changed expression levels of an endogenous cdc27a geneand/or may result from changed expression levels of a cdc27a genepreviously introduced into a plant. Similarly, changed levels and/oractivity of a CDC27A protein may be due to changed expression of anendogenous cdc27a nucleic acid/gene and/or due to changed expression ofa cdc27a nucleic acid/gene previously introduced into a plant.Increasing or decreasing expression of a gene/nucleic acid and/orincreasing or decreasing activity and/or levels of a gene product may beeffected, for example, by chemical means and/or recombinant means.

Advantageously, increase or decrease of expression of a cdc27a nucleicacid and/or increase or decrease of activity and/or levels of a CDC27Aprotein may be effected by chemical means, i.e. by exogenous applicationof one or more compounds or elements capable of increasing or decreasingactivity and/or levels of the CDC27A protein and/or capable ofincreasing or decreasing expression of a cdc27a nucleic acid/gene. Theterm “exogenous application” as defined herein is taken to mean thecontacting or administering of a suitable compound or element to plantcells, tissues, organs or to the whole organism. The compound or elementmay be exogenously applied to a plant in a form suitable for plantuptake (such as through application to the soil for uptake via theroots, or in the case of some plants by applying directly to the leaves,for example by spraying). The exogenous application may take place onwild-type plants or on transgenic plants that have previously beentransformed with a cdc27a nucleic acid/gene or another transgene.

Suitable compounds or elements include CDC27A proteins or cdc27a nucleicacids. Similarly, homologues, derivatives or active fragments of CDC27Aproteins and/or portions or sequences capable of hybridizing with acdc27a nucleic acid may also be used. The exogenous application ofcompounds or elements capable of increasing or decreasing levels offactors that directly or indirectly activate or inactivate a CDC27Aprotein will also be suitable in practising the invention. Also includedare antibodies that can recognise or mimic the function of cdc27Aproteins. Such antibodies may comprise “plantibodies”, single chainantibodies, IgG antibodies and heavy chain camel antibodies, as well asfragments thereof. Additionally or alternatively, the resultant effectmay also be achieved by the exogenous application of an interactingprotein or activator or an inhibitor of the cdc27a gene/gene product.Additionally or alternatively, the compound or element may be amutagenic substance, such as a chemical selected from any one or moreof: N-nitroso-N-ethylurea, ethylene imine, ethyl methanesulphonate anddiethyl sulphate. Mutagenesis may also be achieved by exposure toionising radiation, such as X-rays or gamma-rays or ultraviolet light.Methods for introducing mutations and for testing the effect ofmutations (such as by monitoring gene expression and/or proteinactivity) are well known in the art.

Therefore, according to one aspect of the present invention, there isprovided a method for changing development of a plant, comprisingexogenous application of one or more compounds or elements capable ofincreasing or decreasing expression of a cdc27a gene and/or capable ofincreasing or decreasing activity and/or levels of a CDC27A protein.

Additionally or alternatively, and according to a preferred embodimentof the present invention, increase or decrease of expression of a cdc27anucleic acid and/or increase or decrease of activity and/or levels of aCDC27A protein may be effected by recombinant means. Such recombinantmeans may comprise a direct and/or indirect approach for increase ordecrease of expression of a nucleic acid and/or for increase or decreaseof the activity and/or levels of a protein.

Therefore there is provided by the present invention, a method to changeplant development, comprising increasing or decreasing cdc27a geneexpression and/or CDC27A protein levels and/or CDC27A protein activity,which increase or decrease may be effected by recombinant means and/orby chemical means.

The cdc27a gene or the CDC27A protein in a plant may be wild type, i.e.a native or endogenous nucleic acid or polypeptide. Alternatively, itmay be a nucleic acid derived from the same or another species, whichgene is introduced as a transgene, for example by transformation. Thistransgene may be substantially changed from its native form incomposition and/or genomic environment through deliberate humanmanipulation.

An indirect recombinant approach may comprise for example introducing,into a plant, a nucleic acid capable of increasing or decreasingactivity and/or levels of the protein in question (a CDC27A protein)and/or capable of increasing or decreasing expression of the gene inquestion (a cdc27a gene). Examples of such nucleic acids to beintroduced into a plant, are nucleic acids encoding transcriptionfactors or activators or inhibitors that bind to the promoter of acdc27a gene or that interact with a CDC27A protein. Methods to testthese types of interactions and methods for isolating nucleic acidsencoding such interactors include yeast one-hybrid or a yeast two-hybridscreens.

Also encompassed by an indirect approach for increasing or decreasingactivity and/or levels of a CDC27A protein and/or expression of a cdc27agene, is the provision of, or the inhibition or stimulation ofregulatory sequences that drive expression of the native cdc27a gene orof the cdc27a transgene. Such regulatory sequences may be introducedinto a plant. For example, the nucleic acid introduced into the plant isa promoter, capable of driving the expression of an endogenous cdc27agene.

A further indirect approach for increasing or decreasing activity and/orlevels and/or expression of a cdc27a gene or protein in a plant,encompasses increased or decreased levels in a plant of a factor able tointeract with CDC27A. Such factors may include ligands of CDC27A.Therefore, the present invention provides a method for changingdevelopment of a plant, when compared to the corresponding wild-ypeplants, comprising increasing or decreasing expression of a gene codingfor a protein which is a natural ligand of a CDC27A. Furthermore, thepresent invention also provides a method for changing development of aplant relative to corresponding wild-type plants, comprising increasingor decreasing expression of a gene coding for a protein which is anatural target/substrate of a CDC27A.

A direct and more preferred approach for changing development of aplant, comprises introducing into a plant a cdc27a nucleic acid, or aportion thereof or sequences capable of hybridising therewith, whichnucleic acid preferably encodes a CDC27A protein or a homologue,derivative or active fragment thereof. The nucleic acid may beintroduced into a plant by, for example, transformation.

According to one preferred aspect of the present invention, there isprovided a method for changing plant development, a nucleic acidsequence capable of increasing or decreasing expression of a cdc27a geneand/or capable of increasing or decreasing activity and/or levels of aCDC27A protein. Further preferably such nucleic acid sequence is acdc27a nucleic acid.

As mentioned above the nucleic acid to be used in the methods of thepresent invention can be wild type (native or endogenous).Alternatively, the nucleic acid may be derived from another species,which gene is introduced into the plant as a transgene, for example bytransformation. The nucleic acid may thus be derived (either directly orindirectly (if subsequently modified)) from any source provided that thenucleic acid, when expressed in a plant, leads to increased or decreasedexpression of a cdc27a nucleic acid/gene or increased or decreasedactivity and/or levels of a CDC27A protein. The nucleic acid may beisolated from a microbial source, such as bacteria, yeast or fungi, orfrom a plant, algae, insect, or animal (including human) source. Thisnucleic acid may be substantially changed from its native form incomposition and/or genomic environment through deliberate humanmanipulation. The nucleic acid sequence is preferably a homologousnucleic acid sequence, i.e. a nucleic acid sequence obtained from aplant, whether from the same plant species or different. The nucleicacid may be isolated from a dicotyledonous species, preferably from thefamily Brassicaceae, further preferably from Arabidopsis thaliana. Morepreferably, the nucleic acid is as represented by SEQ ID NO: 1 or aportion thereof or a nucleic acid capable of hybridising therewith or isa nucleic acid encoding an amino acid represented by SEQ ID NO: 2 or ahomologue derivative or active fragment thereof, such as a homologuehaving at least 47%, 48%, 49, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% 98%, 99% sequence identity with SEQ ID NO 2.

Although the invention has been exemplified with a cdc27a according toSEQ ID NO: 1, and corresponding amino acids according to SEQ ID NO: 2,it would be apparent to a person skilled in the art that the methodsaccording to the invention may also be practised using variant nucleicacids and variant amino acids, such as the ones defined hereinafter.

Therefore, taken in a broad context, the term “cdc27a” protein/nucleicacid also encompasses variant nucleic acids and variant amino acidssuitable for practicing the methods according to the invention.Preferably, variant nucleic acids and variant amino acids suitable forpracticing the methods according to the invention include those fallingwithin the definition of a “cdc27a”, meaning that upon construction of aphylogenetic tree, such as the one depicted in FIG. 7, the variantsequences of interest would tend to cluster around cdc27aproteins/genes. Further preferred cdc27a variants cluster around thecdc27a protein of Arabidopsis rather than around the cdc27b protein ofArabidopsis. In case of variants of particular plants for which nodistinction between cdc27A or cdc27B can be made, preferred variants maycluster around a separate group of cdc27 proteins each of which mayrepresent a unique cdc27 protein in the genome of said plant. Examplesof such cdc27 proteins are monocots cdc27 proteins such as representedby SEQ ID NO 6 (rice), SEQ ID NO 8 (sugar cane), SEQ ID NO 10 (maize)and SEQ ID NO 12 (wheat). These cdc27 proteins are also useful for themethods of the present invention. Such a phylogenetic tree can beconstrued with amino acid sequences or with nucleic acid sequences. Aperson skilled in the art could readily determine whether any nucleicacid sequence or protein sequence in question falls within thedefinition of a “cdc27a” using known techniques and software for themaking of such phylogenetic trees, such as a GCG, EBI or CLUSTALpackage, or Align X, using default parameters. Upon construction of sucha phylogenetic tree, sequences clustering in the cdc27a group will beconsidered to fall within the definition of a “cdc27a” as used hereinand will therefore be useful in performing the methods of the invention.

Suitable variant nucleic acid and amino acid sequences useful inpractising the method according to the invention, include:

-   -   (i) Functional portions of a cdc27a nucleic acid/gene;    -   (ii) Sequences capable of hybridising with a cdc27a nucleic        acid/gene;    -   (iii) Alternative splice variants of a cdc27a nucleic acid/gene;    -   (iv) Allelic variants of a cdc27a nucleic acid/gene;    -   (v) Homologues, derivatives and active fragments of a cdc27a        protein;

The term cdc27a nucleic acid/gene, as defined herein, also encompasses acomplement of SEQ ID NO 1 and also to corresponding RNA, DNA, cDNA orgenomic DNA. The cdc27a may be synthesized in whole or in part, it maybe double-strand nucleic acid or single-stranded nucleic acid. Also thisterm encompasses a variant of the gene due to the degeneracy of thegenetic code and variants that are interrupted by one or moreintervening sequences.

An example of a variant cdc27a nucleic acid/gene is a functional portionof a cdc27a nucleic acid/gene. The methods according to the inventionmay advantageously be practised using functional portions of a cdc27a. Afunctional portion refers to a piece of DNA derived or prepared from anoriginal (larger) DNA molecule, which DNA portion, when introduced andexpressed in a plant, gives plants having changed development. Theportion may comprise many genes, with or without additional controlelements or may contain spacer sequences. The portion may be made bymaking one or more deletions and/or truncations to the nucleic acidsequence. Techniques for introducing truncations and deletions into anucleic acid are well known in the art. Portions suitable for use in themethods according to the invention may readily be determined usingroutine techniques, such as by assaying for CDC27A activity and/or byfollowing the methods described in the Examples section by simplysubstituting the sequence used in the actual Example with the portion tobe tested for functionality.

Methods for assaying the activity of a CDC27A protein may comprise

-   -   1. Optionally, a first step of expressing the CDC27A encoding        gene,    -   2. making extracts of the host cell (and optionally purify the        CDC27A protein) and than,    -   3. use it in biological assays in comparison with a wild-type        CDC27A cell extract (or purified protein).

Such biological assay may involve a test in response to hormones andsugar and a comparison of the RT-PCR profile of the investigated proteinwith the profiles of CDC27A.

Another test to investigate the functionality of a CDC27A protein (or afragment, a homologue or derivative thereof) is a yeast complementationassay, wherein the gene/protein under investigation is introduced in ayeast cell missing its natural cdc27 gene and/or protein. Subsequentlyit is checked if these yeast cell are able to form colonies normally andif they are capable of normal DNA synthesis. Such a yeastcomplementation assay has been described by Blilou et al. (Genes Dev.2002 16(19): 2566-75). In brief, to investigate whether the CDC27Aprotein can act as a component of the APC, the full-size cDNA can becloned in a yeast vector with a thiamine-repressible promoter andtransformed into an S. pombe nuc2^(ts) strain. The cdc27A expressionshould at least partially rescue the nuc2 phenotype at the restrictivetemperature, and reproducibly restore growth to higher density comparedwith the empty vector control.

Another assay to test the functionality of a cdc27 protein is a “pulldown” experiment. CDC27 is part of a multiprotein APC complex, bybinding to specific proteins within this complex. To confirm that thisprotein is indeed implicated in this structure, the tandem affinitypurification (TAP) method can be used. The TAP is a tool that allowsrapid purification under native conditions of complexes, even whenexpressed at their natural level. The TAP method requires fusion of theTAP tag, either N- or C-terminally, to the target protein of interest,for example CDC27. By successive elution from affinity columns for thetags, high specific purification of the complex can be obtained. Afterfinal elution step of the purified complex, the identification ofproteins interacting with the given target protein is done via massspectrometry. (Puig et al, (2001) The tandem affinity purification (TAP)method: a general procedure of protein complex purification. Methods24(3):218-29; Rigaut et al. (1999), A generic protein purificationmethod for protein complex characterization and proteome exploration.Nat Biotechnol. 17(10):1030-2).

An example of a further variant cdc27a nucleic acid is a sequence thatis capable of hybridising to a cdc27a. Advantageously, the methodsaccording to the present invention may also be practised using sequencescapable of hybridising to a cdc27a, particularly a cdc27a as representedby any one of SEQ ID NO: 1 or SEQ ID NO: 3, which hybridising sequencesare preferably those falling within the definition of a “cdc27a”,meaning that upon construction of a phylogenetic tree, such as the onedepicted in FIG. 7, the hybridising sequence would be one that tends tocluster around the cdc27a's. Hybridising sequences suitable for use inthe methods according to the invention may readily be determined usingroutine techniques, such as by assaying for CDC27A activity and/or byfollowing the methods described in the Examples section by simplysubstituting the sequence used in the actual Example with thehybridising sequence.

The term “hybridisation” as defined herein is a process whereinsubstantially homologous complementary nucleotide sequences anneal toeach other. The hybridisation process can occur entirely in solution,i.e. both complementary nucleic acids are in solution. Tools inmolecular biology relying on such a process include the polymerase chainreaction (PCR; and all methods based thereon), subtractivehybridisation, random primer extension, nuclease S1 mapping, primerextension, reverse transcription, cDNA synthesis, differential displayof RNAs, and DNA sequence determination. The hybridisation process canalso occur with one of the complementary nucleic acids immobilised to amatrix such as magnetic beads, Sepharose beads or any other resin. Toolsin molecular biology relying on such a process include the isolation ofpoly (A+) mRNA. The hybridisation process can furthermore occur with oneof the complementary nucleic acids immobilised to a solid support suchas a nitro-cellulose or nylon membrane or immobilised by e.g.photolithography to e.g. a siliceous glass support (the latter known asnucleic acid arrays or microarrays or as nucleic acid chips). Tools inmolecular biology relying on such a process include RNA and DNA gel blotanalysis, colony hybridisation, plaque hybridisation, in situhybridisation and microarray hybridisation. In order to allowhybridisation to occur, the nucleic acid molecules are generallythermally or chemically denatured to melt a double strand into twosingle strands and/or to remove hairpins or other secondary structuresfrom single stranded nucleic acids. The stringency of hybridisation isinfluenced by conditions such as temperature, salt concentration andhybridisation buffer composition. High stringency conditions forhybridisation include high temperature and/or low salt concentration(salts include NaCl and Na₃-citrate) and/or the inclusion of formamidein the hybridisation buffer and/or lowering the concentration ofcompounds such as SDS (detergent) in the hybridisation buffer and/orexclusion of compounds such as dextran sulphate or polyethylene glycol(promoting molecular crowding) from the hybridisation buffer.Conventional hybridisation conditions are described in, for example,Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition ColdSpring Harbor Laboratory Press, CSH, New York, but the skilled craftsmanwill appreciate that numerous different hybridisation conditions can bedesigned in function of the known or the expected homology and/or lengthof the nucleic acid sequence. Sufficiently low stringency hybridisationconditions are particularly preferred (at least in the first instance)to isolate nucleic acids heterologous to the DNA sequences of theinvention defined supra. An example of low stringency conditions is4-6×SSC/0.1-0.5% w/v SDS at 37-45° C. for 2-3 hours. Depending on thesource and concentration of the nucleic acid involved in thehybridisation, alternative conditions of stringency may be employed,such as medium stringency conditions. Examples of medium stringencyconditions include 1-4×SSC/0.25% w/v SDS at ≧45° C. for 2-3 hours. Anexample of high stringency conditions includes 0.1-1×SSC/0.1% w/v SDS at60° C. for 1-3 hours. The skilled man will be aware of variousparameters which may be altered during hybridisation and washing andwhich will either maintain or change the stringency conditions. Thestringency conditions may start low and be progressively increased untilthere is provided a hybridising cdc27a nucleic acid, as definedhereinabove. Elements contributing to heterology include allelism,degeneration of the genetic code and differences in preferred codonusage.

Another example of a variant cdc27a is an alternative splice variant ofa cdc27a. The methods according to the present invention may also bepractised using an alternative splice variant of a cdc27a nucleicacid/gene. The term “alternative splice variant” as used hereinencompasses variants of a nucleic acid in which selected introns and/orexons have been excised, replaced or added. Such splice variants may befound in nature or can be manmade using techniques well known in theart. A splice variant useful in the methods according to the inventionis preferably a “cdc27a”, meaning that upon construction of aphylogenetic tree, such as the one depicted in FIG. 7, the splicevariant of interest would be one tending to cluster around the cdc27a'srather than around any of the other CDK groups. Preferably, the splicevariant is a splice variant of the sequence represented by any of SEQ IDNO: 1 or SEQ ID NO: 3. Splice variants suitable for use in the methodsaccording to the invention may readily be determined using routinetechniques, such as by assaying for CDC27A activity and/or by followingthe methods described in the Examples section by simply substituting thesequence used in the actual Example with the splice variant.

Another example of a variant cdc27a is an allelic variant.Advantageously, the methods according to the present invention may alsobe practised using allelic variants of a cdc27a nucleic acid, preferablyan allelic variant of a sequence represented by any of SEQ ID NO: 1 orSEQ ID NO: 3. Allelic variants exist in nature and encompassed withinthe methods of the present invention is the use of these isolatednatural alleles in the methods according to the invention. Allelicvariants encompass Single Nucleotide Polymorphisms (SNPs), as well asSmall Insertion/Deletion Polymorphisms (INDELs). The size of INDELs isusually less than 100 bp). SNPs and INDELs form the largest set ofsequence variants in naturally occurring polymorphic strains of mostorganisms. The allelic variants useful in the methods according to theinvention are preferably “cdc27a”, meaning that upon construction of aphylogenetic tree, such as the one depicted in FIG. 7, the allelicvariant of interest would tend to cluster around the cdc27a's. Allelicvariants suitable for use in the methods according to the invention mayreadily be determined using routine techniques, such as by assaying forCDC27A activity and/or by following the methods described in theExamples section by simply substituting the sequence used in the actualExample with the allelic variant.

Accordingly, the present invention provides a method for changing plantdevelopment, wherein the cdc27a nucleic acid sequence is a splicevariant of a cdc27a nucleic acid sequence or wherein said CDC27A proteinis encoded by a splice variant or wherein the cdc27a nucleic acidsequence is an allelic variant of a cdc27a nucleic acid sequence orwherein said CDC27A protein is encoded by an allelic variant.

Examples of variant CDC27A amino acids include homologues, derivativesand active fragments of a CDC27A protein. Advantageously, the methodsaccording to the present invention may also be practised usinghomologues, derivatives or active fragments of a CDC27A, preferablyusing homologues, derivatives or active fragments of a CDC27A's asrepresented by any one of SEQ ID NO: 2 or SEQ ID NO: 4.

“Homologues” of a CDC27A protein encompass peptides, oligopeptides,polypeptides, proteins and enzymes having amino acid substitutions,deletions and/or insertions relative to the unchanged protein inquestion and having similar biological and functional activity as theunchanged protein from which they are derived. To produce suchhomologues, amino acids of the protein may be replaced by other aminoacids having similar properties (such as similar hydrophobicity,hydrophilicity, antigenicity, propensity to form or break α-helicalstructures or β-sheet structures). Conservative substitution tables arewell known in the art (see for example Creighton (1984) Proteins. W.H.Freeman and Company).

The homologues useful in the methods according to the invention have apercentage identity to SEQ ID NO 2 or 4 equal to value lying between 47%and 99.99%.

The homologues useful in the method according to the invention have atleast 47%, 48%, 49% or 50% sequence identity or similarity (functionalidentity) to the unchanged protein, alternatively at least 60% sequenceidentity or similarity to an unchanged protein, alternatively at least70% sequence identity or similarity to an unchanged protein. Typically,the homologues have at least 80% sequence identity or similarity to anunchanged protein, preferably at least 85%, 86%, 87%, 88%, 98% sequenceidentity or similarity, further preferably at least 90%, 91%, 92%, 93%,94% sequence identity or similarity to an unchanged protein, mostpreferably at least 95%, 96%, 97%, 98% or 99% sequence identity orsimilarity to an unchanged protein.

The percentage of identity can be calculated by using an alignmentprogram well known in the art. For example, the percentage of identitycan be calculated using the program GAP, or needle (EMBOSS package) orstretcher (EMBOSS package) or the program align X, as a module of thevector NTI suite 5.5 software package, using the standard parameters(for example GAP penalty 5, GAP opening penalty 15, GAP extensionpenalty 6.6).

The homologues useful in the methods according to the invention arepreferably “cdc27a”, meaning that upon construction of a phylogenetictree, such as the one depicted in FIG. 7, the homologue of interestwould tend to cluster around the CDC27A. A preferred CDC27A homologuehas more sequence identity with the Arabidosis thaliana cdc27A protein(SEQ ID NO 1) than to another Arabidopsis thaliana protein (for examplethe Arabidopsis thaliana CDC27B protein, genbank accession numberCAD31951). The sequence identity between AtCDC27A and AtCDC27B is 46.8%when calculated with the alignX program as mentioned above. Therefore,preferred homologues useful in the methods of the present invention arehomologues having more than 47% sequence identity with AtCDC27A.Homologues suitable for use in the methods according to the inventionmay readily be determined using routine techniques, such as by assayingfor CDC27A activity and/or by following the methods described in theExamples section by simply substituting the sequence used in the actualExample with the homologous sequence.

Methods for the search and identification of CDC27A homologues or DNAsequences encoding a CDC27A homologue, would be well within the realm ofpersons skilled in the art. Such methods, involve screening sequencedatabases with the sequences as provided by the present invention in SEQID NO 1 and 2, preferably a computer readable format of the nucleicacids of the present invention. This sequence information is availablefor example in public databases, that include but are not limited toGenbank (http://www.ncbi.nlm.nih.gov/web/Genbank), the EuropeanMolecular Biology Laboratory Nucleic Acid Sequence Database (EMBL)(http:/w.ebi.ac.uk/ebi-docs/embl-db.html) or versions thereof or theMIPS database (http://mips.gsf.de/). Different search algorithms andsoftware for the alignment and comparison of sequences are well known inthe art. Such software includes software include GAP, BESTFIT, BLAST,FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch (J.Mol. Biol. 48: 443-453, 1970) to find the alignment of two completesequences that maximises the number of matches and minimises the numberof gaps. The BLAST algorithm calculates percentage sequence identity andperforms a statistical analysis of the similarity between the twosequences. The suite of programs referred to as BLAST programs has 5different implementations: three designed for nucleotide sequencequeries (BLASTN, BLASTX, and TBLASTX) and two designed for proteinsequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology:76-80, 1994; Birren et al., Genome Analysis, 1: 543, 1997). The softwarefor performing BLAST analysis is publicly available through the NationalCentre for Biotechnology Information.

Homologues of SEQ ID NO 2 can be found in many prokaryotic andeukaryotic organisms. The closest homologues are found in the plantkingdom. For example, partial cdc27a nucleic acids were isolate fromrice (SEQ ID NO 5) encoding a rice cdc27 homologue (SEQ ID NO 6), fromsugar cane (SEQ ID NO 7 and 8), from maize (SEQ ID NO 9 and 10), fromsorghum (SEQ ID NO 11 and 12) and from wheat (SEQ ID NO 13 and 14).

As more genomes are being sequenced, it is expected that many moreCDC27A homologues shall be identifiable.

These above-mentioned analysis for comparing sequences, ispreferentially done on a full-length sequence or alternatively can bebased on a comparison of certain regions such as conserved domains.

The identification of such domains, would also be well within the realmof a person skilled in the art and involves for example, a computerreadable format of the nucleic acids of the present invention, the useof alignment software programs and the use of publicly availableinformation on protein domains, conserved motifs and boxes. This proteindomain information is available in the PRODOM(http://www.biochem.ucl.ac.uk/bsm/dbbrowser/jj/prodomsrchjj.html), PIR(http://pir.georgetown.edu/) or pFAM (http://pfam.wustl.edu/) database.Sequence analysis programs designed for motif searching can be used foridentification of fragments, regions and conserved domains as mentionedabove. Preferred computer programs would include but are not limited toMEME, SIGNALSCAN, and GENESCAN. A MEME algorithm (Version 2.2) can befound in version 10.0 of the GCG package; or on the Internet sitehttp://www.sdsc.edu/MEME/meme. SIGNALSCAN version 4.0 information isavailable on the Internet sitehttp://biosci.cbs.umn.edu/software/sigscan.html. GENESCAN can be foundon the Internet site http://gnomic.stanford.edu/GENESCANW.html.

More particularly preferred cdc27A homologues have the conserved domainsas described in WO0102430, which text on domains is incorporated hereinby reference. More particularly they comprise TRP domain repeats(Interpro database accession number IPR001440 repeat domain) and/or theso-called cdc27/NUC-like domain (prodom database accession numberPD555428) or a domain which aligns with these domains when scanned withthe above mentioned software for domain identification.

Two special forms of homology, orthologous and paralogous, areevolutionary concepts used to describe ancestral relationships of genes.The term “paralogous” relates to gene-duplications within the genome ofa species. The term “orthologous” relates to homologous genes indifferent organisms due to ancestral relationship. The term “homologues”as used herein also encompasses paralogues and orthologues and areuseful proteins in the methods according to the invention.

Another variant of CDC27A useful in the methods of the present inventionis a derivative of CDC27A. The term “derivatives” refers to peptides,oligopeptides, polypeptides, proteins and enzymes which may comprisesubstitutions, deletions or additions of naturally and non-naturallyoccurring amino acid residues compared to the amino acid sequence of anaturally-occurring form of the protein, for example, as presented inSEQ ID NO: 2. “Derivatives” of a CDC27A protein encompass peptides,oligopeptides, polypeptides, proteins and enzymes which may comprisenaturally occurring changed, glycosylated, acylated or non-naturallyoccurring amino acid residues compared to the amino acid sequence of anaturally-occurring form of the polypeptide. A derivative may alsocomprise one or more non-amino acid substituents compared to the aminoacid sequence from which it is derived, for example a reporter moleculeor other ligand, covalently or non-covalently bound to the amino acidsequence such as, for example, a reporter molecule which is bound tofacilitate its detection, and non-naturally occurring amino acidresidues relative to the amino acid sequence of a naturally-occurringprotein.

“Substitutional variants” of a protein are those in which at least oneresidue in an amino acid sequence has been removed and a differentresidue inserted in its place. Amino acid substitutions are typically ofsingle residues, but may be clustered depending upon functionalconstraints placed upon the polypeptide; insertions will usually be ofthe order of about 1 to 10 amino acid residues, and deletions will rangefrom about 1 to 20 residues. Preferably, amino acid substitutionscomprise conservative amino acid substitutions.

“Insertional variants” of a protein are those in which one or more aminoacid residues are introduced into a predetermined site in a protein.Insertions can comprise amino-terminal and/or carboxy-terminal fusionsas well as intra-sequence insertions of single or multiple amino acids.Generally, insertions within the amino acid sequence will be smallerthan amino- or carboxy-terminal fusions, of the order of about 1 to 10residues. Examples of amino- or carboxy-terminal fusion proteins orpeptides include the binding domain or activation domain of atranscriptional activator as used in the yeast two-hybrid system, phagecoat proteins, (histidine)₆-tag, glutathione S-transferase-tag, proteinA, maltose-binding protein, dihydrofolate reductase, Tag-100 epitope,c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HAepitope, protein C epitope and VSV epitope.

“Deletion variants” of a protein are characterised by the removal of oneor more amino acids from the protein. Amino acid variants of a proteinmay readily be made using peptide synthetic techniques well known in theart, such as solid phase peptide synthesis and the like, or byrecombinant DNA manipulations. Methods for the manipulation of DNAsequences to produce substitution, insertion or deletion variants of aprotein are well known in the art. For example, techniques for makingsubstitution mutations at predetermined sites in DNA are well known tothose skilled in the art and include M13 mutagenesis, T7-Gen in vitromutagenesis (USB, Cleveland, Ohio), QuickChange Site Directedmutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directedmutagenesis or other site-directed mutagenesis protocols.

Another variant of CDC27A useful in the methods of the present inventionis an active fragment of CDC27A. “Active fragments” of a CDC27A proteinencompasses contiguous amino acid residues of a CDC27A protein, whichresidues retain similar biological and/or functional activity to thenaturally occurring protein. For example, useful fragments comprise atleast 10 contiguous amino acid residues of a CDC27A protein. Otherpreferred fragments are fragments of the CDC27A protein starting at thesecond or third or further internal methionin residues. These fragmentsoriginate from protein translation, starting at internal ATG codons.

According to a preferred aspect of the present invention, enhanced orincreased expression of a cdc27a nucleic acid in a plant or plant partis envisaged. Methods for obtaining increased expression of genes orgene products are well documented in the art and include, for example,overexpression driven by a (strong) promoter, the use of transcriptionenhancers or translation enhancers. The term overexpression as usedherein means any form of expression that is additional to the originalwild-type expression level. Preferably the nucleic acid to be introducedinto the plant and/or the nucleic acid that is to be overexpressed inthe plants is in the sense direction with respect to the promoter towhich it is operably linked.

Accordingly, a preferred embodiment of the present invention provides amethod to change development in a plant, comprising introducing, into aplant, a nucleic acid sequence capable of increasing or decreasingexpression of a cdc27a gene and/or capable of increasing or decreasingactivity and/or level of a CDC27A protein in the sense orientationrelative to control element to which it is operably linked.

Alternatively and/or additionally, increased expression of a CDC27Aencoding gene or increased activities and/or levels of a CDC27A proteinin a plant cell, is achieved by mutagenesis. For example these mutationscan be responsible for the changed control of the cdc27a gene, resultingin more expression of the gene, relative to the wild-type gene.Mutations can also cause conformational changes in a protein, resultingin more activity and/or levels of the CDC27A protein.

Since accelerated rate of development has been demonstrated via plantsoverexpressing the cdc27a gene, there is envisaged by the presentinvention a method for delaying development comprising downregulation ofexpression of a cdc27a gene or downregulation of levels and/or activityof a CDC27A protein. Also methods encompassing downregulation of CDC27Acan be used to decrease the number or the size of organs or to delayflowering. Therefore, according to a further aspect of the invention,decreased expression of a cdc27a nucleic acid or decreased activityand/or level of a CDC27A is envisaged.

Examples of decreasing or downregulation of expression are welldocumented in the art and include, for example, downregulation ofexpression by anti-sense techniques, RNAi techniques, small interferenceRNAs (siRNAs), microRNA (mRNA), etc. Therefore according to a particularaspect of the invention, there is provided a method for changingdevelopment of plants, including technologies that are based on forexample the synthesis of antisense transcripts, complementary to themRNA of a cdc27a gene.

Another method for downregulation of gene expression or gene silencingcomprises use of ribozymes, for example as described in WO9400012(Atkins et al.), WO9503404 (Lenee et al.), WO0000619 (Nikolau et al.),WO9713865 (Ulvskov et al.) and WO9738116 (Scott et al.).

Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion) or by gene silencingstrategies as described among others in the documents WO9836083(Baulcombe and Angell), WO9853083 (Grierson et al.), WO9915682(Baulcombe et al.) or WO9953050 (Waterhouse et al.).

Expression of an endogenous gene may also be reduced if the endogenousgene contains a mutation. Such a mutant gene may be isolated andintroduced into the same or different plant species in order to obtainplants having changed development. Also dominant negative mutants of acdc27a nucleic acid can be introduced in the cell to decrease thelevel/and or activity of the endogenous CDC27a protein.

Other methods to decrease the expression of a cdc27a nucleic acid and/oractivity and/or level of CDC27A proteins in a cell encompass for examplethe mechanisms of transcriptional gene silencing, such as themethylation of the cdc27a promoter.

Another mechanism to downregulate levels and/or activity of a CDC27Aprotein in a plant encompasses the mechanism of co-suppression.Increasing or decreasing gene expression (whether by a direct orindirect approach) encompasses changed transcript levels of that gene.Changed transcript levels can be sufficient to induce certain phenotypiceffects, for example via the mechanism of cosuppression. Here theoverall effect of expression of a transgene is that there is lessactivity in the cell of the protein encoded by a native gene havinghomology to the introduced transgene. Cosuppression is accomplished bythe addition of coding sequences or parts thereof in a sense orientationinto the cell. Therefore, according to one aspect of the presentinvention, the development of a plant may be changed by introducing intoa plant an additional copy (in full or in part) of a cdc27a gene alreadypresent in a host plant. The additional gene may silence the endogenousgene, giving rise to a phenomenon known as co-suppression.

Genetic constructs aimed at silencing gene expression may comprise thecdc27a nucleotide sequence or one at least a portion thereof in a senseand/or antisense orientation relative to the promoter sequence.Preferably the portions comprises at least 21 contiguous nucleic acid ofa sequence to be downregulated. Also, sense or antisense copies of atleast part of the endogenous gene in the form of direct or invertedrepeats may be utilised in the methods according to the invention. Thedevelopment of plants may also be changed by introducing into a plant atleast part of an antisense version of the nucleotide sequencerepresented, for example, by SEQ ID NO: 1. It should be clear that partof the nucleic acid (a portion) could also achieve the desired result.Homologous anti-sense genes are preferred, homologous genes being plantgenes, preferably plant genes from the same plant species in which thesilencing construct is introduced.

The expression of a cdc27a gene can be investigated by northern orSouthern blot analysis of cell extracts. The levels of CDC27A protein inthe cell can be investigated via Western blot analysis of cell extracts.

The activity of a CDC27A protein can be investigated by making extractsof a cell (and optionally purify the CDC27A protein) and than use it inbiological assays in comparison with a wild-type CDC27A cell extract (orpurified protein). Such biological assay may involve a test in responseto hormones and sugar and a comparison of the RT-PCR profile of theinvestigated protein with the profiles of CDC27A.

Another test to investigate the functionality of a CDC27A protein (or afragment, a homologue or derivative thereof) is a yeast complementationassay, wherein the gene/protein under investigation is introduced in ayeast cell missing its natural cdc27 gene and/or protein. Subsequentlyit is checked if these yeast cell are able to form colonies normally andif they are capable of normal DNA synthesis. Such a yeastcomplementation assay has been described by Blilou et al. (Genes Dev.2002 16(19): 2566-75). In brief, to investigate whether the CDC27Aprotein can act as a component of the APC, the full-size cDNA can becloned in a yeast vector with a thiamine-repressible promoter andtransformed into an S. pombe nuc2^(ts) strain. The cdc27A expressionshould at least partially rescue the nuc2 phenotype at the restrictivetemperature, and reproducibly restore growth to higher density comparedwith the empty vector control.

Another assay to test the functionality of a cdc27 protein is a “pulldown” experiment. CDC27 is part of a multiprotein APC complex, bybinding to specific proteins within this complex. To confirm that thisprotein is indeed implicated in this structure, the tandem affinitypurification (TAP) method can be used. The TAP is a tool that allowsrapid purification under native conditions of complexes, even whenexpressed at their natural level. The TAP method requires fusion of theTAP tag, either N- or C-terminally, to the target protein of interest,for example CDC27. By successive elution from affinity columns for thetags, high specific purification of the complex can be obtained. Afterfinal elution step of the purified complex, the identification ofproteins interacting with the given target protein is done via massspectrometry. (Puig et al, (2001) The tandem affinity purification (TAP)method: a general procedure of protein complex purification. Methods24(3):218-29; Rigaut et al. (1999), A generic protein purificationmethod for protein complex characterization and proteome exploration.Nat Biotechnol. 17(10):1030-2).

According to second embodiment of the present invention, geneticconstructs and vectors to facilitate introduction and/or expression ofthe nucleotide sequences useful in the methods according to theinvention are provided. Therefore, according to the second emboddiment,the present invention provides a genetic construct comprising:

-   -   (i) a nucleic acid sequence capable of increasing or decreasing        expression of a cdc27a nucleic acid and/or capable of increasing        or decreasing the activity and/or level of a CDC27A protein;    -   (ii) one or more control sequences capable of regulating        expression of the nucleic acid sequence of (i); and optionally    -   (iii) a transcription termination sequence.

According to the methods of the present invention, such a vector isintroduced into a plant or plant part.

Constructs useful in the methods according to the present invention maybe constructed using recombinant DNA technology well known to personsskilled in the art. The gene constructs may be inserted into vectors,which may be commercially available, suitable for transforming intoplants and suitable for expression of the gene of interest in thetransformed cells. The genetic construct can be an expression vectorwherein said nucleic acid sequence is operably linked to one or morecontrol sequences allowing expression in prokaryotic and/or eukaryotichost cells.

The nucleic acid according to (i) is advantageously any of theaforementioned nucleic acids, preferably a cdc27a nucleic acid, mostpreferably a cdc27a nucleic acod according to SEQ ID NO 1 or 3. Theconstruct sequence of (ii) is preferably a constitutive promoter, forexample a CaMV35S or GOS2 promoter

The methods according to the present invention may also be practised byintroducing into a plant at least a part of a (natural or artificial)chromosome (such as a Bacterial Artificial Chromosome (BAC)), whichchromosome contains at least a cdc27a gene/nucleic acid, optionallytogether with one or more related gene family members. Therefore,according to a further aspect of the present invention, there isprovided a method for changing plant development by introducing into aplant at least a part of a chromosome comprising at least a cdc27agene/nucleic, which cdc27a gene/nucleic is preferably one represented byany one of SEQ ID NO: 1 or SEQ ID NO: 3.

According to a preferred embodiment of the invention, the geneticconstruct is an expression vector designed to overexpress the nucleicacid sequence. The nucleic acid sequence capable of increasing ordecreasing expression of a cdc27a nucleic acid and/or activity and/orlevel of a CDC27A protein itself may be a cdc27a nucleic acid or ahomologue, derivative or active fragment thereof, such as any of thenucleic acid sequences described hereinbefore. A preferred nucleic acidsequence is the sequence represented by SEQ ID NO: 1 or 3 or a portionthereof or sequences capable of hybridising therewith or a nucleic acidsequence encoding a sequence represented by SEQ ID NO: 2 or 4 or ahomologue, derivative or active fragment thereof. Preferably, thisnucleic acid is cloned in the sense orientation relative to the controlsequence.

Plants are transformed with a vector comprising the sequence of interest(i.e., the nucleic acid sequence capable of increasing or decreasingexpression of cdc27a nucleic acid), which sequence is operably linked toone or more control sequences (at least a promoter). The terms“regulatory element”, “control sequence” and “promoter” are all usedherein interchangeably and are to be taken in a broad context to referto regulatory nucleic acid sequences capable of effecting expression ofthe sequences to which they are ligated (i.e. operably linked).Encompassed by the aforementioned terms are transcriptional regulatorysequences derived from a classical eukaryotic genomic gene (includingthe TATA box which is required for accurate transcription initiation,with or without a CCAAT box sequence) and additional regulatory elements(i.e. upstream activating sequences, enhancers and silencers) whichalter gene expression in response to developmental and/or externalstimuli, or in a tissue-specific manner. Also included within the termis a transcriptional regulatory sequence of a classical prokaryoticgene, in which case it may include a −35 box sequence and/or −10 boxtranscriptional regulatory sequences. The term “regulatory element” alsoencompasses a synthetic fusion molecule or derivative which confers,activates or enhances expression of a nucleic acid molecule in a cell,tissue or organ. The term “operably linked” as used herein refers to afunctional linkage between the promoter sequence and the gene ofinterest, such that the promoter sequence is able to initiatetranscription of the gene of interest.

Advantageously, any type of promoter may be used to drive expression ofthe nucleic acid sequence depending on the desired outcome. For example,a meristem-specific promoter, such as the rnr (ribonucleotidereductase), cdc2a promoter and the cyc07 promoter. Also seed-specificpromoter, such as p2S2, pPROLAMIN, pOLEOSIN could be selected. Analeurone-specific promoter may be selected. An inflorescence-specificpromoter, such as pLEAFY, may also be utilised. To produce male-sterileplants one would need an anther specific promoter. One could also choosea petal-specific promoter. If the desired outcome would be to changedevelopment in particular organs, then the choice of the promoter woulddepend on the organ to be changed. For example, use of a root-specificpromoter would lead to phenotypic alteration of the root. This would beparticularly important where it is the root itself that is the desiredend product; such crops include sugar beet, turnip, carrot, and potato.A fruit-specific promoter may be used to modify, for example, thestrength of the outer skin of the fruit or to increase the size of thefruit. A green tissue-specific promoter may be used to influence thephenotype pf the leaf. A cell wall-specific promoter may be used toincrease the rigidity of the cell wall, thereby increasing pathogenresistance. An anther-specific promoter may be used to producemale-sterile plants. A vascular-specific promoter may be used toincrease transport from leaves to seeds. A nodule-specific promoter maybe used to increase the nitrogen fixing capabilities of a plant, therebyincreasing the nutrient levels in a plant. A stress-inducible promotermay also be used to drive expression of a nucleic acid during conditionsof stress. A stress inducible promoter such as the water stress inducedpromoter WSI18, the drought stress induced Trg-31 promoter, the ABArelated promoter rab21 or any other promoter which is induced under aparticular stress condition such as temperature stress (cold, freezing,heat) or osmotic stress, or drought stress or oxidative stress or bioticstress can be used to drive expression of a cdc27a gene.

Preferably, the nucleic acid sequence capable of increasing ordecreasing expression of a cdc27a gene is operably linked to aconstitutive promoter. The term “constitutive” as defined herein refersto a promoter that is expressed predominantly in at least one tissue ororgan, and predominantly at any life stage of the plant. Preferably thepromoter is expressed predominantly in most tissues or organs of theplant, most preferably throughout the whole plant. Preferably, theconstitutive promoter is a CaMV35s promoter or GOS2 promoter, or apromoter of similar strength and/or a promoter with a similar expressionpattern. Similar strength and/or similar expression pattern can beanalysed for example by coupling the promoters to a reporter gene andcheck the function of the reporter gene in tissues of the plant. Onesuitable reporter gene is beta-glucuronidase and the colorimetric GUSstaining to visualize the reporter gene activity in a plant tissue iswell known to a person skilled in the art. Examples of otherconstitutive promoters are presented in Table 1, which promoters orderivatives thereof are useful in performing the methods of the presentinvention. TABLE 1 EXEMPLARY CONSTITUTIVE PROMOTERS FOR USE IN THEPERFORMANCE OF THE PRESENT INVENTION EXPRESSION GENE SOURCE PATTERNREFERENCE Actin constitutive McElroy et al, Plant Cell, 2: 163- 171,1990 CAMV 35S constitutive Odell et al, Nature, 313: 810-812, 1985 CaMV19S constitutive Nilsson et al., Physiol. Plant. 100: 456-462, 1997 GOS2constitutive de Pater et al, Plant J Nov; 2(6): 837-44, 1992 ubiquitinconstitutive Christensen et al, Plant Mol. Biol. 18: 675-689, 1992 riceconstitutive Buchholz et al, Plant Mol Biol. cyclophilin 25(5): 837-43,1994 maize H3 constitutive Lepetit et al, Mol. Gen. Genet. histone 231:276-285, 1992 actin 2 constitutive An et al, Plant J. 10(1); 107-121,1996

Optionally, one or more terminator sequences may also be used in theconstruct introduced into a plant. The term “terminator” encompasses acontrol sequence which is a DNA sequence at the end of a transcriptionalunit which signals 3′ processing and polyadenylation of a primarytranscript and termination of transcription. Additional regulatoryelements may include transcriptional as well as translational enhancers.Those skilled in the art will be aware of terminator and enhancersequences, which may be suitable for use in performing the invention.Such sequences would be known or may readily be obtained by a personskilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence which is required for maintenance and/orreplication in a specific cell type. One example is when a geneticconstruct is required to be maintained in a bacterial cell as anepisomal genetic element (e.g. plasmid or cosmid molecule). Preferredorigins of replication include, but are not limited to, the f1-ori andcolE1.

The genetic construct may optionally comprise a selectable marker gene.As used herein, the term “selectable marker gene” includes any gene,which confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with a nucleic acid construct of theinvention. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptIIencoding neomycin phosphotransferase capable of phosphorylating neomycinand kanamycin, or hpt encoding hygromycin phosphotransferase capable ofphosphorylating hygromycin), to herbicides (for example bar whichprovides resistance to Basta; aroA or gox providing resistance againstglyphosate), or genes that provide a metabolic trait (such as manA thatallows plants to use mannose as sole carbon source). Visual marker genesresult in the formation of colour (for example beta-glucuronidase, GUS),luminescence (such as luciferase) or fluorescence (Green FluorescentProtein, GFP, and derivatives thereof. Further examples of suitableselectable marker genes include the ampicillin resistance (Ampr),tetracycline resistance gene (Tcr), bacterial kanamycin resistance gene(Kanr), phosphinothricin resistance gene, and the chloramphenicolacetyltransferase (CAT) gene, amongst others

In a preferred embodiment, the genetic construct as mentioned above,comprises a cdc27a nucleic acid in the sense orientation coupled to apromoter that is preferably a constitutive promoter, such as for examplethe GOS2 promoter. Therefore according to another aspect of theinvention, there is provided an isolated nucleic acid, comprising anexpression cassette, comprising at least a part of a nucleic acidsequence depicted in SEQ ID NO 1 or 3, or the complementary strandthereof; operably linked to at least a part of a constitutive promoter.

According to a third embodiment of the present invention, there isprovided a method for the production of a plant having changeddevelopment, comprising increasing or decreasing expression and oractivity and/or levels in a plant of a cdc27a nucleic acid or CDC27Aprotein. According to a particular embodiment, the present inventionprovides a method for the production of transgenic plants having changedgrowth characteristics, which method comprises:

-   -   (i) introducing into a plant or plant part a nucleic acid or a        portion thereof or sequences capable of hybridising therewith,        which nucleic acid is capable of increasing or decreasing        expression of a cdc27a gene and/or capable of increasing or        decreasing the activity and/or levels of a CDC27A, preferably        wherein said nucleic acid encodes a CDC27A protein or a        homologue, derivative or active fragment thereof;    -   (ii) cultivating the plant cell under conditions promoting        regeneration and mature plant growth.

The nucleic acid of (i) may advantageously be any of the aforementionednucleic acids, preferably a cdc27a nucleic acid, most preferably acdc27a nucleic acid according to SEQ ID NO 1 or 3. The nucleic acid ispreferably operably linked to a constitutive promoter such as a CaMV35Sor GOS2 promoter.

The protein itself and/or the nucleic acid itself may be introduceddirectly into a plant cell or into the plant itself (includingintroduction into a tissue, organ or any other part of the plant).According to a preferred feature of the present invention, the nucleicacid is preferably introduced into a plant by transformation.

The term “transformation” as referred to herein encompasses the transferof an exogenous polynucleotide into a host cell, irrespective of themethod used for transfer. Plant tissue capable of subsequent clonalpropagation, whether by organogenesis or embryogenesis, may betransformed with a genetic construct of the present invention and awhole plant regenerated therefrom. The particular tissue chosen willvary depending on the clonal propagation systems available for, and bestsuited to, the particular species being transformed. Exemplary tissuetargets include leaf disks, pollen, embryos, cotyledons, hypocotyls,megagametophytes, callus tissue, existing meristematic tissue (e.g.,apical meristem, axillary buds, and root meristems), and inducedmeristem tissue (e.g. cotyledon meristem and hypocotyl meristem). Thepolynucleotide may be transiently or stably introduced into a host celland may be maintained non-integrated, for example, as a plasmid.Alternatively and preferably, the transgene may be stably integratedinto the host genome. The resulting transformed plant cell can then beused to regenerate a transformed plant in a manner known to personsskilled in the art.

Transformation of a plant species is now a fairly routine technique.Advantageously, any of several transformation methods may be used tointroduce the gene of interest into a suitable ancestor cell.Transformation methods include the use of liposomes, electroporation,chemicals that increase free DNA uptake, injection of the DNA directlyinto the plant, particle gun bombardment, transformation using virusesor pollen and microprojection. Methods may be selected from thecalcium/polyethylene glycol method for protoplasts (Krens, F. A. et al.,1882, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol.8, 363-373); electroporation of protoplasts (Shillito R. D. et al., 1985Bio/Technol 3, 1099-1102); microinjection into plant material (CrosswayA. et al., 1986, Mol. Gen Genet 202, 179-185); DNA or RNA-coatedparticle bombardment (Klein T. M. et al., 1987, Nature 327, 70)infection with (non-integrative) viruses and the like.

Transgenic rice plants expressing a cdc27a gene are preferably producedvia Agrobacterium-mediated transformation using any of the well knownmethods for rice transformation, such as described in any of thefollowing: published European patent application EP 1198985 A1, Aldemitaand Hodges (Planta, 199, 612-617, 1996); Chan et al. (Plant Mol. Biol.22 (3) 491-506, 1993), Hiei et al. (Plant J. 6 (2) 271-282, 1994), whichdisclosures are incorporated by reference herein as if fully set forth.In the case of corn transformation, the preferred method is as describedin either Ishida et al. (Nat. Biotechnol. 1996 June; 14(6): 745-50) orFrame et al. (Plant Physiol. 2002 May; 129(1): 13-22), which disclosuresare incorporated by reference herein as if fully set forth.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant.

Following DNA transfer and regeneration, putatively transformed plantsmay be evaluated, for instance using Southern analysis, for the presenceof the gene of interest, copy number and/or genomic organisation.Alternatively or additionally, expression levels of the newly introducedDNA may be monitored using Northern and/or Western analysis, bothtechniques being well known to persons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention also encompasses plants obtainable by the methodsaccording to the present invention. The present invention thereforeprovides plants obtainable by the method according to the presentinvention, which plants have changed development, when compared to thewild-type plants and which plants have increased or decreased CDC27Aprotein activity and/or levels and/or increased or decreased expressionof a cdc27a nucleic acid.

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedin the parent by the methods according to the invention. The inventionaccordingly also includes host cells containing an isolated nucleic acidmolecule encoding a CDC27A protein. Preferred host cells according tothe invention are plant cells. The invention also extends to harvestableparts of a plant such as but not limited to seeds, leaves, fruits,flowers, stem cultures, stem, rhizomes, roots, tubers and bulbs.

Preferably said plants are transformed with a CDC27A encoding gene underthe control of a constitutive promoter and more preferably the plants ofthe present invention carry an expression cassette comprising at least apart of cdc27A and at least a part of a constitutive promoter asmentioned hereinabove. The host cells, plants or the plant parts of thepresent invention can be identified by the presence of higher expressionof a cdc27a gene and/or or a higher level and/or activity of a cdc27Aprotein. Further, particular plants of the present invention arerecognizable by the presence of a cdc27a transgene or part thereofgenetically coupled to a constitutive promoter, preferably to a CaMV35Spromoter or to a GOS2 promoter or any promoter as described hereinabove,or at least a part thereof.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,roots (including tubers), and plant cells, tissues and organs. The term“plant” also therefore encompasses suspension cultures, embryos,meristematic regions, callus tissue, leaves, gametophytes, sporophytes,pollen, and microspores. Plants that are particularly useful in themethods of the invention include all plants which belong to thesuperfamily Viridiplantae, in particular monocotyledonous anddicotyledonous plants including a fodder or forage legume, ornamentalplant, food crop, tree, or shrub selected from the list comprisingAcacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathisaustralis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachisspp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaeaplurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkeaafricana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camelliasinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens,Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermummopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumisspp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeriajaponica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergiamonetaria, Davallia divancata, Desmodium spp., Dicksonia squarosa,Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum,Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestisspp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulaliavillosa, Fagopyrum spp., Feioa sellowiana, Fragaria spp., Flemingia spp,Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypertheliadissoluta, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago sativa, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Omithopus spp., Oryza spp.,Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp.,Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp.,Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthriafleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria,Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercusspp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis,Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubusspp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata,Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor,Spinacia spp., Sporobolus fimbiratus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusselssprouts, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugar beet, sugar cane, sunflower, tomato, squash tea, trees.Alternatively algae and other non-Viridiplantae can be used for themethods of the present invention. Preferably the plant according to thepresent invention is a crop plant selected from rice, maize, wheat,barley, soybean, sunflower, canola, sugarcane, alfalfa, millet,leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, popular andcotton. Further preferably, the plant according to the present inventionis a monocotyledonous plant, most preferably a cereal.

The term development as used herein means the process to reach maturityand reproductive stage, involving differentiation of the cells and organformation.

A change in development means a change in time to reach maturity as wellas a change in developmental characteristics which are manifestations ofdevelopment such as differentiation and/or organ formation.

Advantageously, the present invention provides a method to change plantdevelopment, wherein the changed development is preferably selected fromchanged differentiation, changed rate of development, changed organformation, changed organ size and/or number, and changed reproductivecharacteristics, relative to the wild-type plants.

Further preferably, the changed differentiation is accelerateddifferentiation and/or the changed rate of development is acceleratedrate of development and/or the changed organ formation is acceleratedorgan formation and/or the changed organ size and/or number is increasedorgans size and/or number and/or the changed reproductive characteristicis early flowering and increased number of flowers and/or seeds.

The plants according to the present invention show accelerateddifferentiation. This feature is particularly advantageous foraccelerating crop production.

The plants according to the present invention show accelerated rate ofdevelopment. Therefore, in addition to the accelerated rate ofdifferentiation (which is typically a phenomenon for cells leaving themeristem), the faster rate of development is manifested in more than onepart of a plant and throughout the life of the plant. The effects of acdc27A may also be more pronounced in more mature stages of the plant.

The plants according to the present invention, show accelerated organformation. More particularly, the formation of leaves, flowers and seedsis accelerated. These features are particularly interesting forhorticultural applications as well as agriculture, in particular forcrop plants harvested for their green biomass (grasses), flowers(cotton) or their seeds (cereals).

The plants according to the present invention have increased size andnumber, more particularly bigger leaves, longer leaves, wider leaves,more leaves, longer stem, more flowers, more seed pods, more seeds.

The methods of the present invention clearly change the appearance ormorphology of a plant, including any one or more structural features orcombination of structural features thereof. Therefore the plantsaccording to the present invention have changed architecture whencompared to the wild-type plants. Other structural features, which maybe altered by the methods of the present invention include shape, size,number, position, texture, arrangement, and pattern of any cell, tissueor organ or groups of cells, tissues or organs of a plant, including theroot, leaf, shoot, stem or tiller, petiole, trichome, flower,inflorescence (for monocotyledonous and dicotyledonous plants),panicles, petal, stigma, style, stamen, pollen, ovule, seed, embryo,endosperm, seed coat, aleurone, fibre, cambium, wood, heartwood,parenchyma, aerenchyma, sieve elements, phloem or vascular tissue,amongst others.

The plants according to the present invention have changed reproductivecharacteristics. The term “reproductive characteristic” as used hereinencompasses the characteristics, which are involved in flowering time,time to reach the flowering stage and the reproductive organs (such asfor example flowers and flowers parts and the seeds). More particularly,the plants of the present invention show early flowering, relative tocorresponding wild-type plants. The characteristic of early flowering isparticularly favourable for any crop plant, since the life cycle (i.e.cycling time) of the plants is reduced and harvesting can take placesooner. Consequently the agricultural land is available sooner forfurther crops. Also land, which is normally not available foragriculture because of the too short growing season, may now becomeaccessible for the plants of the present invention.

The plans according to the present invention have more flowers and seedsand therefore have increased yield.

The term “increased yield” encompasses an increase in biomass in one ormore parts of a plant relative to the biomass of corresponding wild-typeplants. The term also encompasses an increase in seed yield, whichincludes an increase in the biomass of the seed (seed weight) and/or anincrease in the number of (filled) seeds and/or in the size of the seedsand/or an increase in seed volume, each relative to correspondingwild-type plants. An increase in seed size and/or volume may alsoinfluence the composition of seeds. An increase in seed yield could bedue to an increase in the number and/or size of flowers. An increase inyield might also increase the harvest index, which is expressed as aratio of the total biomass over the yield of harvestable parts, such asseeds.

Accordingly, a particular embodiment of the present invention relates toa method to increase seed yield and/or to increase harvest index of acereal. The methods of the present invention are therefore particularlyfavourable to be applied to crop plants, preferably seed crops andcereals, because the methods of the present invention are used toincrease the seed yield and harvest index of the plant. Therefore, themethods of the present invention are particularly useful for plantsgrown for harvest of seeds, such as cereals, rapeseed, sunflower,leguminosae (e.g. soybean, pea, bean) flax, lupinus, canola etc. . . .

Additionally or alternatively, the plants according to the inventionhave more leaves and bigger stems. Therefore, the methods of the presentinvention are additionally and/or alternatively particularly favourableto crops grown for the green tissue and/or grown for the above groundbiomass. The methods of the present invention are particularly usefulfor increasing leaf size and number of grasses and forage crops (such asforage maize, clover, medicago alfalfa etc.). The methods of the presentinvention are also particularly useful for increasing the stem size oftrees (for paper and pulp industry) and sugar cane.

The present invention also relates to use of a cdc27a nucleic acid andto the use of portions thereof or nucleic acids hybridising therewith inchanging development, differentiation and organ formation of plants. Thepresent invention also relates to use of a CDC27A protein and to the useof homologues, derivatives and active fragments thereof in changingdevelopment, differentiation and organ formation of plants. The nucleicacid sequence is preferably as represented by SEQ ID NO: 1 or 3 or aportion thereof or sequences capable of hybridising therewith or is anamino acid sequence represented by SEQ ID NO: 2 or 4 or a homologue,derivative or active fragment thereof.

The present invention also relates to the use of a cdc27a nucleic acidand to the use of portions thereof or nucleic acids hybridisingtherewith and to the use of the CDC27A protein itself and of homologues,derivatives and active fragments thereof as regulators of plantdevelopment. The nucleic acid sequences hereinbefore described (andportions of the same and sequences capable of hybridising with the same)and the amino acid sequences hereinbefore described (and homologues,derivatives and active fragments of the same) are useful in changingdevelopment of plants, as hereinbefore described. The sequences wouldtherefore find use as regulator of such processes, such as regulators ofrate of development, rate of organ formation, regulator of organ numberand size, or regulator of reproductive characteristics, such asflowering time, number of flowers and seeds.

The present invention also provides a composition comprising a proteinrepresented by any of the aforementioned amino acid sequences orhomologues, derivatives or active fragments thereof for the use as aregulator of developmental processes and characteristics, such asmentioned hereinabove.

Conversely, the sequences according to the present invention may also beinteresting targets for agrochemical compounds, such as herbicides orgrowth stimulators. Accordingly, the present invention encompasses useof the aforementioned nucleic acid sequences (or a portion of the sameor sequences capable of hybridising with the same) or an amino acidsequence as hereinbefore described (or homologues, derivatives andactive fragments of the same) as targets for an agrochemical compound,such as a herbicide or a growth stimulator.

According to another aspect of the present invention, advantage may betaken of the nucleotide sequence capable of increasing or decreasingexpression of a cdc27a nucleic acid in breeding programmes. The nucleicacid sequence may be on a chromosome, or a part thereof, comprising atleast the cdc27a nucleic acid sequence and preferably also one or morerelated family members. In an example of such a breeding programme, aDNA marker is identified which may be genetically linked to a genecapable of increasing or decreasing expression of a cdc27a nucleic acidin a plant, which gene may be a gene encoding the CDC27A protein itselfor any other gene which may directly or indirectly influence expressionof the cdc27a gene and/or activity of the CDC27A protein itself. ThisDNA marker may then be used in breeding programs to select plants havingchanged development.

Further the use of allelic variants as described herein-above areparticularly useful for conventional breeding programmes, such as inmarker-assisted breeding, which is also encompassed by the presentinvention. Such breeding programmes sometimes require the introductionof allelic variations in the plants by mutagenic treatment of a plant.One suitable mutagenic method is EMS mutagenesis. Identification ofallelic variants then may take place by, for example, PCR. Tilling ispreferred for identifying allelic variants. This is followed by aselection step for selection of superior allelic variants of the CDC27Asequence and which give rise to changed development in a plant.Selection, according to the method of the present invention, istypically carried out by monitoring development, differentiation andorgan formation of plants containing different allelic variants of theCDC27A sequence, for example, different allelic variants of SEQ ID NO: 1or of a CDC27A orthologue in that plant. Monitoring growth performancecan be done in a greenhouse or in the field. Further optional stepsinclude crossing plants, in which the superior allelic variant wasidentified, with another plant. This could be used, for example, to makea combination of interesting phenotypic features. Therefore, mutationsin the cdc27a gene may occur naturally, and may form the basis of theselection of plants showing accelerated rate of development, increasedorgan size and/or number, and/or early flowering.

Accordingly, as another aspect of the invention, there is provided amethod for the selection of plants having changed development, whichmethod is based on the selection of better-performing allelic variantsof the CDC27A sequence relative to the wild-type allele, and which giverise to changed development in a plant.

According to another aspect of the invention, there is also provided amethod for generating plants having changed plant development, whencompared to the corresponding wild-type plant, which method comprisesthe steps of:

a. Growing a plant with increased or decreased expression of a cdc27anucleic acid sequence and/or having increased or decreased levels and/oractivity of a CDC27A protein, when compared to the wild-type plant, and

b. Crossing said plant of (a) with a plant of interest; and

c. Producing progeny of the cross, and optionally,

d. selecting the progeny with said changed development.

Alternatively, the cdc27a gene itself can be used as a (genetic) markerto detect the presence or absence of a desired trait, or QuantitativeTrait Locus (QTLs). In this application of the present invention thegene encoding CDC27A is genetically linked to the desired trait, andtypically the phenotypes caused by the gene encoding a CDC27A aremonitored in order to breed and select plants with the desired trait.This desired trait or QTL, may comprise a single gene or a cluster oflinked genes that affect the desired trait.

In molecular biology it is standard practice to select upon transfectionor transformation those individuals (or groups of individuals, such asbacterial or yeast colonies or phage plaques or eukaryotic cell clones)that are effectively transfected or transformed with the desired geneticconstruct. Typically these selection procedures are based on thepresence of a selectable or screenable marker in the transfected geneticconstruct, to distinguish the positive individuals easily from thenegative individuals. Therefore, the cdc27a gene can also be used forthese purposes, since introduction of this gene into a host cell resultsin changed development of said host cell.

The methods according to the present invention may also be practised byco-expression of a cdc27a gene in a plant with at least one other genethat cooperates with the cdc27a gene. Co-expression may be effected bycloning the genes under the control of a plant expressible promoter in aplant expressible vector and introducing the expression vector(s) into aplant cell using Agrobacterium-mediated plant transformation.

The methods according to the present invention result in plants havingchanged development, as described hereinbefore. These advantageousdevelopmental characteristics may also be combined with othereconomically advantageous traits, such as further yield-enhancingtraits, tolerance to various stresses, traits increasing or decreasingvarious architectural features and/or biochemical and/or physiologicalfeatures. Accordingly, the methods of the present invention can also beused in so-called “gene stacking” procedures.

Also the present invention encompasses a food product derived from anyof the plants produced by the methods of the present invention. Furtherthe invention also refers to the use of a product derived from any ofthe plants according to the present invention in animal feed and in foodor in the production procedures thereof.

In a particular embodiment of the invention the plants with improveddevelopmental characteristics are used to produce industrial enzymesand/or pharmaceuticals. The production of such enzymes orpharmaceuticals in plants is aimed at high accumulation of the desiredproducts in a particular and easy to harvest plant tissues, for exampleaccumulation in the leaves and/or in the seeds. The plants of thepresent invention have bigger stems, bigger leaves, more leaves, moreflowers and/or more seeds, and therefore are capable of producing moreindustrial enzymes and/or pharmaceuticals in these tissues, moreparticularly in their green biomass and/or in their seeds. Accordingly,the present invention also provides a method for the production ofenzymes and/or pharmaceuticals, which method comprises the increasing ordecreasing of expression of a cdc27a gene or the increasing ordecreasing of activity and/or level of a CDC27A protein. Further theinvention relates to the use of plants according to the invention forthe production of industrial enzymes and pharmaceuticals and theinvention extends to the industrial enzymes and pharmaceuticals producesaccording to these methods.

DESCRIPTION OF THE FIGURES

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1 is a schematic representation of the construct used fortransforming the plants of the present invention.

FIG. 2 illustrates that leaves develop faster in cdc27a transgenicplants (35S13.3/1) compared to control plants (SR1). Number 1 to 6correspond to the leaves as they grow on the stem of the plant, meaningthat leaf 1 is the leaf developed in the juvenile plant and leaf 6 beingthe most recently developed leaf, i.e. a leaf developed when the sameplant is in a more mature stage.

FIG. 3 illustrates transgenic plants transformed with the construct35S::cdc27a (positions 1 to 4) and a non transgenic control plant(position 5). The plants are photographed at the time when the firstplants start to flower. The transgenic plants as can be seen in theillustration are taller.

FIG. 4 is a graphical illustration indicating the length of the leaves 6(A), and 7 (B) of cdc27a transgenic plants compared to the control line(SR1).

FIG. 5 is a graphical illustration indicating the width of the leaves 6(A) and 7 (B) of cdc27a transgenic plants compared to the control line(SR1).

FIG. 6 is the nucleic acid sequence and protein sequence of theArabidopsis thaliana cdc27A proteins useful for the methods of thepresent invention.

FIG. 7 is a phylogenetic tree showing the structural and evolutionalrelationship between A-type cdc27 proteins and B-type cdc27 proteins ofArabidopsis thaliana. The tree was construed using the software Align Xas part of the VNTi suite 5.5 software package. As an outgroup; theprotein sequence of Atcdc20 was used. The sequences are annotated bytheir Genbank accession number or by the International publicationnumber of the patent application in which they are described.

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone.

Unless otherwise stated, recombinant DNA techniques are performedaccording to standard protocols described in Sambrook (2001) MolecularCloning: a laboratory manual, 3rd Edition Cold Spring Harbor LaboratoryPress, CSH, New York; or in Volumes 1 and 2 of Ausubel et al. (1988),Current Protocols in Molecular Biology, Current Protocols. Standardmaterials and methods for plant molecular work are described in PlantMolecular Biology Labfase (1993) by R. D. D. Croy, published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications(UK).

Example 1 Cloning of the cdc27a Gene

To express constitutively the Arabidopsis cdc27a cDNA in transgenicplants, the full-length cdc27a gene was isolated and cloned as follows.To introduce suitable restriction sites in the cdc27a cDNA, a PCRreaction has been carried out using oligonucleotides containing NcoI andBamHI restriction sites. The resulting fragment was restricted with the2 enzymes (NcoI and BamHI). The cdc27a open reading frame was ligated inthe PH35S vector (Hemerly et al. EMBO J. 14, 3925-3936), which wasopened with the Nco1 and BamH1 restriction enzymes. The resultingexpression cassette contained the CaMV35S promoter, the Atcdc27a geneand the NOS terminator (see FIG. 1). Subsequenlty, this plasmid wasdigested with EcoRI, filled in with Klenow enzyme, and then cut withSalI to release the expression cassette containing the 35S promoter, theCDC27 reading frame, and the NOS terminator. This fragment was cloned inthe PGSV4 plasmid in the SalI and ScaI sites. The resulting expressionplasmid was introduced in Agrobacterium tumefasciens C58.

Example 2 Transformation of Tobacco Cells with the 35S::cdc27a Construct

Tobacco plants were transformed with the Agrobacterium strain asmentioned in Example 1 comprising the CDC27A expression vector. Forintroduction of the cdc27a gene into tobacco plants, the leaf diskmethod was used (Horsch et al., 1985; A simple and general method fortransferring genes into plants Science 227 1229-1231). From thesetransformed leaf disks, T0 plants were regenerated and allowed to sedseeds (T1 seeds). These T1 seeds were germinated in medium containingkanamycin to determine the number of loci of the transgene. Plants witha 3 to 1 relation of kanamycin resistant to susceptible seedlings werechosen to produce seeds in order to obtain homozygous plants.

Example 3 cdc27a Transgenic Tobacco Plants Develop Faster

Wild type tobacco plants and transgenic tobacco plants weresimultaneously grown in the same growth conditions. The leaves oftransgenics and non-transgenic plants were cut off, sorted by age (1 isthe oldest leaf and 6 is the newest developed leaf on the stem) andphotographed (see FIG. 2).

The upper line designated SR1 shows the leaves of a non-transgeniccontrol plant and the lower line designated 35S13.3/1, shows the leavesof a transgenic plant transformed with the 35S::cdc27a construct. Thiscomparative picture of the leaves at the same age of both plants,illustrates that the transgenic leaves are bigger. Therefore it can beconcluded that the transgenic leaves develop faster. It was alsoobserved that the transgenic seedlings produce leaves earlier than thewild-type control plants. Therefore it can be concluded that thedevelopmental program in transgenics progresses faster than that of thewild-type plants. The picture of FIG. 2 further illustrates that theeffects of CDC27A on plant development becomes progressively morepronounced as the plant matures. These results illustrate thattransgenic plants have an accelerated rate of development.

Further it was demonstrated that the leaves of cdc27a transgenic plantshad increased leaf length. Wild type tobacco plants and transgenictobacco plants were simultaneously grown in the same growth conditionsand the leaves 6 and 7 were harvested at several days after sowing (seeFIG. 4). This numbering corresponds to the leaf numbering of FIG. 2. Itwas observed that for the recent leaves 6 and 7, the leaves of thetransgenic plants develop quicker (FIGS. 4 A and B respectively). Thisis illustrated by the fact that the transgenic leaves are longer at thesame age as the leaves of wild-type. The transgenic leaf 6 is alreadydeveloped and is 400 mm long on day 45 after sowing, while the leaf 6 ofthe wild-type plant is just being formed. It is illustrated that theleaves 6 and 7 of the transgenic plant lines (1.1, 1.3, 18.1, 25, 3.2)are longer than that of control line SR1 line. The transgenic leaveswere longer than the wild-type leaves at the same age, which age ispreferably before the mature stage.

Further it was demonstrated that the leaves of cdc27a transgenic plantshad increased leaf width. Wild type tobacco plants and transgenictobacco plants were simultaneously grown in the same growth conditionsand the leaves 6 and 7 were harvested and measured at several days aftersowing (see FIG. 5). This numbering corresponds to the leaf numbering ofFIG. 2. It is illustrated that the leaf width of the newly developedleaves 6 and 7 of the transgenic plant lines (1.1, 1.3, 18.1, 25, 3.2)is larger than of the control line SR1.

The transgenic leaves were wider than the wild-type leaves at the sameage, which age is preferably before the mature stage.

Example 4 cdc27a Transgenic Plants Show Increased Biomass

Wild type tobacco plants and transgenic tobacco plants weresimultaneously grown in the same growth conditions and a representativeset of plants were photographed when the first plants reached theflowering stage (see FIG. 3).

At this point, the transgenic plants (numbers 1 to 4 in FIG. 3) as canbe seen in the illustration are taller and have reached a size, that canbe the double or more of the size of non-transgenic plants (number 5 inFIG. 3) grown over the same period of time. Moreover at this timetransgenic plants have produced 18 leaves in the mean versus 12 to 13 inthe wild-type plants. In conclusion, the increased stem size and leafnumber both contribute to the increased total biomass in the cdc27atransgenic plants. The fact that besides leaf size (see example 3) alsostem size and also the number of leaves is increased, further supportsthe finding that CDC27A overexpression accelerates overall vegetativedevelopment.

Example 5 cdc27a Transgenic Plants Show Early Flowering

Wild type tobacco plants and transgenic tobacco plants weresimultaneously grown in the same growth conditions and the plants werephotographed when the transgenic plants started to flower (see FIG. 3).It was observed that the transgenic plants had a reduced period of timeto reach flowering. 4 out of 5 transgenics lines flowered within 127days after sowing while wild-type plants took almost 20 days more (seeTable 2).

The transgenic plants formed an inflorescence with perfectly healthyflowers, with no penalty on the vegetative tissues at the time offlowering, no penalty on the number of flowers or number of seeds (seeTable 3).

This is noteworthy, since the phenotype of early flowering in many casesis associated with reduction in vegetative biomass, a reduction innumber of inflorescence and flowers, and a reduction in seed setting.For example, mutations in the el4 gene result in early flowering and inthese plants, the early flowing phenotype is typically associated withreduced total leaf number (Doyle et al. Nature 2002 419: 74-77).Furthermore, in TFL1 mutant plants which flower early, the earlyflowering phenotype is associated with a penalty on flower structure(Shannon and Meeks-Wagner, 1991 The plant cell 3, 877-892). Also, theearly flowering phenotype of ebs mutants is associated with a reductionof seed dormancy, plant size and fertility (Gomez-Mena et al., 2001, Theplant cell 13, 1011-1024).

The early flowering phenotype demonstrates that CDC27A overexpressionchanged plant development.

Example 6 cdc27a Transgenic Plants at the Time of Flowering are Tallerthan WT Plants at the Time of Flowering

Transgenic plants transformed with CDC27A flower earlier than controlplants (see column flowering time in Table 2). It was further observedthat by the time the transgenic plants have reached the flowering stage,the plants were taller than the non-transgenic control plant at the samestage of flowering (see column plant height at flowering time). Fromthese data it is concluded that the transgenic plants show faster(accelerated rate of development, that they flower early and that theyare bigger in size. These data indicate that cdc27a transgenic plantshave increased biomass.

Example 7 cdc27a Transgenic Plants have More Flowers than WT Plants

At the time of flowering, the flowers were counted from cdc27atransgenic plants and from wild-type plants grown in the sameconditions. Transgenic plants transformed with 35S::cdc27a have moreflowers (see Table 3). Measurements involved five plants of eachtransgenic line and measurements of the control plants involved two SR1plants. These data illustrate that the introduction of CDC27A in plantcan lead to more than a doubling of the amount of flowers. The number ofseed pods is accordingly increases while the size of seed pods was notreduced in the transgenic plants. Therefore, it is envisaged that byusing the methods of the present invention also the number of seeds isincreased. TABLE 2 flowering time, mean Plant height mean at leaf numberat Leaf length/ Line genotype after sowing (in days) * flowering time(cm) * flowering time * width ratio  1.1 homozygous 126.5 +− 11.13 63.8+− 11.77 19.25 +− 1.98   1.87 +− 0.327 ***  1.3 homozygous 123.3 +−16.66 66.3 +− 24.12 17.6 +− 3.75 1.76 +− 0.36 *** 18.8 hemizygous 124.8+− 7.17  59.9 +− 16.70 18.2 +− 1.61 1.69 +− 0.28 *** 25 ** homozygous138.5 +− 20.30 37.87 +− 19.98    17 +− 3.65 1.95 +− 0.27 *** 32homozygous 127.2 +− 7.79  41.1 +− 11.11 16.8 +− 1.28 1.71 +− 0.28 ***SR1 no transgene 147.6 +− 16.30  29 +− 4.6  17.8 +− 1.35 1.78 +− 0.13**** 95% interval of confidence** three of five plants*** Mean ± SD calculated from leaf 6 of five plants 74 days after sowing

TABLE 3 Line 1.1 1.3 18.1 25 32 SR1 Number 23.25 31.33 21.2 14 18.2 12.5of flowers

Example 8 Use of the Invention in Corn

The invention described herein can also be used in maize. To this aim, acdc27a, for example a maize or other ortholog, is cloned under controlof a constitutive promoter operable in maize, in a plant transformationvector suited for Agrobacterium-mediated corn transformation. Methods touse for corn transformation have been described in literature (Ishida etal., Nat Biotechnol. 1996 June;14(6):745-50; Frame et al., PlantPhysiol. 2002 May;129(1):13-22). Transgenic plants made by these methodsare grown in the greenhouse for T1 seed production. Inheritability andcopy number of the transgene are checked by quantitative real-time PCRand Southern blot analysis and expression levels of the transgene aredetermined by reverse PCR and Northern analysis. Transgenic lines withsingle copy insertions of the transgene and with varying levels oftransgene expression are selected for T2 seed production. Progeny seedsare germinated and grown in the greenhouse in conditions well adaptedfor maize (16:8 photoperiod, 26-28° C. daytime temperature and 22-24° C.nighttime temperature) as well under water-deficient,nitrogen-deficient, and excess NaCl conditions. Null segregants from thesame parental line, as well as wild type plants of the same cultivar areused as controls. The progeny plants resulting from the selfing or thecrosses are evaluated on different biomass and developmental parameters,including, stem size, number of leaves, total above ground area, leafgreenness, time to maturity, flowering time, time to flower, ear number,harvesting time. The seeds of these lines are also checked on variousparameters, such as grain size, total grain yield per plant, and grainquality (starch content, protein content and oil content). Lines thatare most significantly improved versus the controls for any of the abovementioned parameters are selected for further field testing andmarker-assisted breeding, with the objective of transferring thefield-validated transgenic traits into commercial germplasm. Methods fortesting maize for growth and yield-related parameters in the field arewell established in the art, as are techniques for introgressingspecific loci (such as transgene containing loci) from one germplasminto another. Corn plants according to the present invention havechanged development, changed rate of development changed organformation, changed organ size and number, and/or changed reproductivecharacteristics, such as early flowering and increased number of flowersand seeds.

Example 9 Use of the Invention in Rice

The invention described herein can also be used in rice. To this aim, acdc27a, for example a rice or other ortholog, is cloned under control ofa constitutive promoter operable in rice, such as for example the GOS2promoter, in a plant transformation vector suited forAgrobacterium-mediated transformation of rice. Such vectors and methodsfor rice transformation have been described in literature. The methodyielded single locus transformants at a rate of over 50% are describedin Aldemita and Hodges, Planta, 199 612-617, 1996; Chan et al., PlantMol. Biol. 22 (3) 491-506, 1993, Hiei et al., Plant J., 6 (2) 271-282,1994) or in EP1198985).

Transgenic plants generated by these rice transformation methods areevaluated for various developmental parameters. More particularly, thetransgenic plants are evaluated and the following parameters aremonitored: increased total above ground biomass, increased plant height,increased number of tillers, increased number of first panicles,increased number of second panicles, increased total number of seeds,increased number of filled seeds, increased total seed yield per plant,increased harvest index, increased thousand kernel weight, increasedTmid, increased T45 or A90, increased A42, changed cycling time or anchanged growth curve, changed flowering time.

Plants with increase rate of development, increased organ formation,increase number and size of organs, reduces flowering time, more flowersand/or more seeds are selected with the objective of transfering thetransgenic traits into commercial germplasm

1. Method to change development of a plant or plant part compared to thewild-type plant or plant part, said method comprising: increasing ordecreasing expression in a plant or plant part of a cdc27a nucleic acidsequence and/or increasing or decreasing levels and/or activity in aplant of a CDC27A protein.
 2. Method according to claim 1, wherein saidincreased or decreased cdc27a expression, CDC27A protein level or CDC27Aprotein activity, is effected by recombinant means and/or by chemicalmeans.
 3. Method according to claim 1, comprising introducing into aplant, a nucleic acid sequence capable of increasing or decreasingexpression of a cdc27a gene and/or capable of increasing or decreasingactivity and/or levels of a CDC27A protein.
 4. Method according to 3,wherein said nucleic acid sequence is a cdc27a nucleic acid.
 5. Methodaccording to claim 4, wherein said nucleic acid is from a dicotyledonousplant.
 6. Method according to claim 3, wherein said nucleic acidsequence is an allelic variant of a cdc27a nucleic acid sequence orwherein said CDC27A protein is encoded by an allelic variant.
 7. Methodaccording to claim 3, wherein said nucleic acid sequence is a splicevariant of a cdc27a nucleic acid sequence or wherein said CDC27A proteinis encoded by a splice variant.
 8. Method according to claim 3, whereinsaid nucleic acid sequence is introduced in a sense direction into aplant.
 9. Method according to claim 3, wherein expression of saidnucleic acid is driven by a constitutive promoter.
 10. Method accordingto claim 1, wherein said changed development is selected from changeddifferentiation, changed rate of development, changed organ formation,changed organ size and/or number, and/or changed reproductivecharacteristics, relative to the wild-type characteristics.
 11. Methodaccording to claim 10, wherein said changed differentiation isaccelerated differentiation or wherein said changed rate of developmentis accelerated rate of development or wherein said changed organformation is accelerated organ formation.
 12. Method according to claim10, wherein said changed organ size and/or number is increased organsize and/or number, such as increased number of leaves, increased numberof flowers, increased number of seeds, increased size of the stem,increased size of the leaf or increased total biomass.
 13. Methodaccording to claim 10, wherein said changed reproductive characteristicis changed flowering characteristic, compared to the wild-type. 14.Method for the production of a transgenic plant having changeddevelopment, compared to a wild-type plant of the same plant species,said method comprising: introducing into a plant, a nucleic acidsequence capable of increasing or decreasing expression of a cdc27a geneand/or capable of increasing or decreasing activity and/or levels of aCDC27A protein; and optionally cultivating the plant cell underconditions promoting regeneration and mature plant growth.
 15. Methodfor generating plants having changed plant development, when compared towild-type plants of the same plant species, said method comprising:Growing a plant with increased or decreased expression of a cdc27anucleic acid sequence and/or having increased or decreased levels and/oractivity of a CDC27A protein, when compared to the wild-type plants, andCrossing said plant of (a) with a plant of interest; and Producingprogeny of the cross, and optionally selecting said progeny with saidchanged development
 16. A method according to claim 1, comprising theintroduction into a plant of a construct comprising, a nucleic acidsequence capable of increasing or decreasing expression of a cdc27anucleic acid and/or capable of increasing or decreasing levels and/oractivity of a CDC27A protein; one or more control sequence capable ofregulating expression of the nucleic acid sequence of (i) in a plant;and optionally a transcription termination sequence.
 17. Plantobtainable by said method according to claim 1, wherein said plant haschanged development, when compared to corresponding wild-type plants ofthe same species.
 18. Plant having changed development when compared tothe corresponding wild-type plant, wherein said plant has in at leastone cell increased or decreased expression of a cdc27a nucleic acidsequence and/or has in at least one cell increased or decreased levelsand/or activity of a CDC27A protein, when compared to a plant of thesame plant species.
 19. Plant according to claim 17, wherein said plantis a monocotyledonous plant, and/or wherein said plant is selected fromrice, maize, wheat, barley, millet, soybean, leguminosae, rapeseed,sunflower, canola, alfalfa, sugarcane, popular, tobacco, and cotton. 20.Plant part, a propagule or progeny from a plant according to claim 17.21. Genetic construct comprising, a nucleic acid sequence capable ofincreasing or decreasing expression of a cdc27a nucleic acid and/orcapable of increasing or decreasing levels and/or activity of a CDC27Aprotein; one or more control sequence capable of regulating expressionof the nucleic acid sequence of (i) in a plant; and optionally atranscription termination sequence.
 22. Genetic construct according toclaim 21, wherein said nucleic acid is a cdc27a nucleic acid from adicotyledonous plant.
 23. Genetic construct according to claim 21,wherein said control sequence is a constitutive promoter or at least apart thereof.
 24. Plant or plant part comprising a genetic constructaccording to claim 21, wherein said plant or plant part has changeddevelopment. 25-29. (canceled)
 30. A food product derived from saidplant according to claim 17 or from a part of said plant.
 31. An animalfeed or food comprising said plant or plant part according to claim 17.32. A method for the production of one or more enzymes orpharmaceuticals, said method comprising: producing said one or moreenzymes or pharmaceuticals with said plant or plant part according toclaim
 17. 33. One or more industrial enzymes a or pharmaceuticalsproduced by using a plant or plant part the method according to claim32.
 34. Plant according to claim 18, wherein said plant is amonocotyledonous plant, and/or wherein said plant is selected from rice,maize, wheat, barley, millet, soybean, leguminosae, rapeseed, sunflower,canola, alfalfa, sugarcane, popular, tobacco, and cotton.
 35. Plantpart, a propagule or progeny from said plant according to claim
 18. 36.A food product derived from said plant according to claim 18 or from apart of said plant.
 37. A food product derived from said plant or plantpart according to claim 24.